Angiogenesis, the process of new blood-vessel growth, plays an essential role in many physiological and pathological processes. It is a multi-step process including endothelial cell activation, proliferation, migration, penetration of extracellular matrix, reorganization of cells into tubules, formation of a lumen, and anastomosis. Typically, angiogenesis is tightly regulated by pro- and anti-angiogenic factors and is crucial for development, reproduction and repair. Vasculogenesis and angiogenesis are down-regulated in the healthy adult and are—except for the organs of the female reproductive system—almost exclusively associated with pathology when angiogenesis is induced by microenvironmental factors (e.g. hypoxia or inflammation). Pathological processes associated with, or induced by, angiogenesis include diseases as diverse as cancer, and inflammatory disorders such as rheumatoid or rheumatic inflammatory disease, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disorders, such as chronic asthma, arterial or post-transplantational atherosclerosis, psoriasis, asthma, infections, obesity, diabetes, endometriosis, ocular neovascularisation, such as retinopathies (including diabetic retinopathy), macular degeneration, thrombosis, hemangioblastoma, and hemangioma (Folkman J., 2007 Nat Rev Drug Discov 6(4):273; Fiedler U. and Augustin H. G. 2006 TRENDS Immun 27(12):552; Li L., et al, 2005 Pediatr Endocrinol Rev. 2(3):399) Although a variety of factors can modulate endothelial cell (EC) responses in vitro, and blood vessel growth in vivo, only vascular endothelial growth factor (VEGF) family members and the angiopoietins are believed to act almost exclusively on vascular ECs. (Yancopoulos G., et al, 2000 Nature 407:242).
The formation of functional vasculature is a complex process requiring spatial and temporal coordination of multiple angiogenic factors, receptors, intracellular signaling pathways and regulatory factors. Without being bound by theory, vascular endothelial growth factors (VEGFs) and angiopoietins (Angs) play complementary roles in this process. The Ang family comprises the ligands Ang1, Ang2, Ang3, and Ang4. Their cognate Tie2/Tek receptor and a closely related orphan receptor, Tie1, are almost exclusively expressed by endothelial cells and hematopoietic stem cells. Tie1 and Tie2 share a similar overall structure consisting of an extracellular domain and an intracellular tyrosine kinase domain. (Shim W. S. N., et al, 2007 Mol Cancer Res 5(7):655; Fiedler U. and Augustin H. G., 2006 TRENDS Immun 27(12):552).
The Angs contain an amino-terminal angiopoietin specific domain, a coiled-coil domain, a linker peptide and a carboxy-terminal fibrinogen homology domain. The fibrinogen homology domain is responsible for receptor binding, the coiled-coil domain is required for dimerization of angiopoietin monomers, and the short amino-terminal region forms ring-like structures that cluster dimers into variable sized multimers necessary for Tie2 activation. (Eklund L. and Olsen B. R, 2006 Exp Cell Res 312:630)
Ang1 is known to form trimers and multimers to homodimerize and induce tyrosine phosphorylation of the Tie2 receptor for intracellular signaling. Dimeric form of Ang1 has been found to inactivate Tie2 receptor, and some isoforms of Ang1 have been reported to negatively regulate Tie2 activation (Shim W. S. N., et al, 2007 Mol Cancer Res 5(7):655). Ang1 binding to the extracellular domain of Tie2 results in receptor dimerization, allowing activation of the kinase domain and autophosphorylation of specific tyrosine residues, acting as docking sites for a number of effectors that couple the activated receptors to the cytoplasmic signaling pathways. Ang1-stimulated Tie2 activation mediates remodeling and stabilization of cell-cell and cell-matrix interactions and plays a role in the recruitment of peri-endothelial mesenchymal cells to the vessels. In addition, Ang1 has anti-permeability and anti-inflammatory functions, and is also critically important in the formation of vascular networks during developmental angiogenesis (Eklund L. and Olsen B. R, 2006 Exp Cell Res 312:630; Shim W. S. N., et al, 2007 Mol Cancer Res 5(7):655).
Ang2 forms dimers to bind to Tie2, but does not induce autophosphorylation. In contrast to Ang1, it is almost exclusively expressed by endothelial cells. Ang2 mRNA is almost undetectable in the quiescent vasculature, however, it is induced dramatically at sites of endothelial cell activation and vascular remodeling. Ang2 expression is induced by various cytokines, including VEGF and fibroblast growth factor (FGF-2), and by microenvironmental factors. Ang2 is upregulated together with VEGF-A at sites of angiogenic sprouting, whereas reduced VEGF-A expression relative to Ang2 is associated with vascular regression. (Eklund L. and Olsen B. R, 2006 Exp Cell Res 312:630; Shim W. S. N., et al, 2007 Mol Cancer Res 5(7):655).
Ang1-mediated Tie2 signaling functions as the default pathway to control vascular quiescence. Ang1 exerts a protective effect on the endothelium and limits its ability to be activated by exogenous cytokines, thus controls vascular homeostasis and endothelial activation. Proper vascular homeostasis is tightly controlled by balanced Tie2 signaling. Ang2 expression is tightly controlled as well. The release of Ang2 results in rapid destabilization of the endothelium. Moreover, Ang2 triggers an inflammatory response by activating the endothelium and inducing permeability. (Fiedler U and Augustin H. G., 2006 TRENDS Immun 27(12):552).
Ang3 and Ang4 are not well studied but are believed to be interspecies orthologues between mouse and human, respectively (Valenzuela D M, et al, 1999 Proc Natl Acad Sci U S A 96:1904-9). The function of Ang3 and Ang4 in angiogenesis is controversial compared with the more established members of the family. Ang3 has been reported to act as antagonist that interfers with Ang1 activation of Tie2 and Akt in tumor growth. However, Ang3 was found to strongly activate mouse Tie2, but not its human counterpart, whereas Ang4 did not display species selectivity in Tie2 activation (Shim W. S. N., et al, 2007 Mol Cancer Res 50:655).
Angiogenic inhibitors are being vigorously pursued. Currently, several angiogenic inhibitors including bevacizumab (Avastin), thalidomide (Thalomid), lenalidomide (Revlimid), ranibizumab (Lucentis), sutinib (Sutent), sorafenib (Nexavar), and pegaptanib (Macugen) are in clinical use. Several angiogenic inhibitors including bevasiranib, AGN-211745, TG-100801, volociximab, ATG-003, relimid, RTP-801i-14, aflibercept, apremilast, INGN-241, angiostatin, endostatin are in clinical trials and many others are in development. The anti-angiogenic compounds developed include monoclonal antibodies or antibody fragments (e.g. bevacizumab, ranibizumab, and volociximab), aptamers (e.g. pegaptanib and E-10030), small-molecules (e.g. thalidomide, ATG-003, TG-100801, pazopanib, vandetanib, lenalidomide, and cediranib), gene therapy (e.g. angistat, advexin, and INGN-241), recombinant proteins (e.g. aflibercept, ABT-828, and replistatin), small interfering RNA (siRNA, e.g. AGN-211745 and bevasiranib), and peptides (e.g. ABT-510, angiostatin, endostatin).
However, as in all technologies in all times, there is an ongoing need for new improved compounds having anti-angiogenic activity.